2,4-dioxo-1,2,3,4-tetrahydropteridine is well known in the art under the name lumazine. Gabriel and Sonn first disclosed in Ber. Deut. Chem. Ges. (1907) 40:4850 making lumazine from pyrazin-bicarboxamide. Timmis in Nature (1949) 164:139 disclosed the synthesis of 1,3-dimethyl-6-phenyllumazine and 1,3-dimethyl-7-phenyl-lumazine by condensing a 6-amino-5-nitroso-pyrimidine with benzaldehyde or methylphenylketone respectively. Zondler et al. in J. Heterocyclic Chem. (1967) 4:124 and Taylor et al. in Heterocycles (1978) 10:37 disclosed 1,3,6-trimethyllumazine and 1,3-dimethyl-6-ethyllumazine. Yoneda and Higuchi in J. Chem. Soc. Perkin (1977) 1336 disclosed the preparation of various 1,3-dimethyl-6-aryllumazines starting from 6-amino-1,3-dimethyl-5-aryliden-aminouracil. Kang et al. in J. Heterocycl. Chem. (1987) 24:597-601 disclosed reacting 5,6-diamino-1,3-dimethyluracil either with propanetrione-1,3-dioxime followed by cyclization to form 1,3-dimethyllumazine-6-carboxaldoxime, or with oximinoacetone followed by cyclization to form 1,3,6-trimethyllumazine, or else with methylglyoxal to form 1,3,7-trimethyllumazine. Both latter compounds may easily, through acid hydrolysis in the presence of formaldehyde, be converted into the corresponding 1,3-dimethyllumazine-carboxaldehydes which, due to their high carbonyl reactivity, may in turn be converted into other lumazine derivatives. Blicke et al. in J.A.C.S (1954) 76:2798-2800 disclosed 1,3-dimethyl-7-aminolumazine, 1,3,6,7-tetramethyllumazine, 1,3-dimethyl-6,7-dihydroxylumazine and 1,3-dimethyl-6,7-diphenylumazine; Pfleiderer in Chem. Ber. (1957) 90:2588 disclosed 1,3-dimethyl-6-hydroxylumazine and 1,3-dimethyl-7-hydroxylumazine; Pfleiderer et al. in Chem. Ber. (1973) 106:3149-3174 disclosed 1,3-dimethyl-6-hydroxy-7-phenyllumazine, 1,3-dimethyl-6-phenyl-7-hydroxylumazine and 1,3-dimethyl-6,7-diisopropylumazine; Hutzenlaub et al. in Chem. Ber. (1973) 106:3203-3215 disclosed 1,3-dimethyl-7-methoxylumazine, 1,3,6-trimethyl-7-hydroxy-lumazine and 1,3,6-trimethyl-7-methoxylumazine; Steppan et al. in Liebigs Ann. Chem. (1982) 2135-2145 disclosed 1,3-dimethyl-6-aminolumazine, 1,3-dimethyl-6-chlorolumazine, 1,3-dimethyl-7-chlorolumazine and 1,3-dimethyl-7-methylaminolumazine; Kasimierczuk et al. in Chem. Ber. (1979) 112:1499-1513 disclosed 1,3-dimethyl-7-mercaptolumazine and 1,3-dimethyl-7-methylthio-lumazine as well as a few substituted 2- or 4-thiolumazines and 2,4-dithiolumazines, starting from substituted 6-amino-2-thiouracil or 6-amino-2,4-dithiouracil; Eisele et al. in Pteridines (1993) 4:178-186 disclosed 1,3,6-trimethyllumazine-7-carboxylic acid and its methyl and ethyl esters. Perez-Rubalcaba et al. in Liebigs Ann. Chem. (1983) 852-859 disclosed substituted 3-methyllumazines wherein one of the 6- and 7-substituents is phenyl whereas the other is chloro. Finally, Weisenfeldt (1987) disclosed a series of tetra-substituted lumazines wherein the 1- and 3-substituents are methyl and one of the 6- and 7-substituents is chloro. Further, Fink et al. in Chem. Berichte (1963) 96:2950-2963, as well as Pfleiderer, Perez-Rubalcaba and Eisele (all cited supra) disclosed bi- and tri-substituted lumazines wherein only one of the 1- and 3-nitrogen atoms is substituted. Interestingly, none of the above-cited substituted lumazines, 2-thiolumazines and 2,4-dithiolumazines was ever said to have any kind of biological activity.
A few other substituted pteridine-2,4-diones (lumazines) are already known in the art as being useful in the preparation of medicines. For instance, U.S. Pat. No. 3,071,587 teaches cyanoethylpteridinediones having central nervous system (hereinafter referred as CNS) activity and anti-depressant properties. WO 94/06431 teaches a 1-methyl-3-(10,11-epoxyundecyl)pteridinedione being able to inhibit IL-1 receptors, decrease proliferation of tumor & other cells, stimulate hematopoeisis, suppress T-cell activation, secretion of antibodies by B-cells and activation of macrophage or endothelial cells by endotoxins, tumor necrosis factor (hereinafter TNF), IL-1 or GM-CSF and enhance resistance of mesenchymal cells to TNF. WO 94/11001 teaches 1-methyl-3-(hydroxy- and dihydroxy-C9-25 alkyl)pteridinediones being able to inhibit lysophosphatidic acid transferase as well as immune or cellular response to stimuli, and therefore can be used to treat tumor progression or invasion, autoimmune diseases, acute allergic reactions mediated by TNF or IL-1, rheumatoid arthritis, osteoarthritis, multiple sclerosis, diabetes, atherosclerosis, restenosis, stroke, HIV infection, inflammatory response, septic shock, CNS and bone diseases. Cottam et al. in J. Med. Chem. (1996) 39 :2-9 and WO 98/52948 both disclose a 1-methyl-3-n-hexyl-6-carboxymethyl-7-carboxymethyl pteridinedione which, although included in a biological evaluation study of inhibitors of TNF-α, was not tested for TNF-α activity. WO 96/20710 teaches substituted pteridinediones which inhibit cellular responses to ceramide metabolites of the sphingomyelin signal transduction pathway, inhibit inflammatory response associated with TNF-α and fibroblast proliferation or UV-induced cutaneous immune suppression and therefore can be used to treat cirrhosis, cell senescence and apoptosis.
Nevertheless, there still is a need in the art for specific and highly therapeutically active compounds, such as, but not limited to, drugs for treating immune and autoimmune disorders, organ and cells transplant rejections, cell proliferative disorders, cardiovascular disorders, disorders of the central nervous system and viral diseases. In particular, there is a need in the art to provide immunosuppressive compounds or antineoplastic drugs or anti-viral drugs which are active in a minor dose in order to replace existing drugs having significant side effects and to decrease treatment costs.
Currently used immunosuppressive drugs include antiproliferative agents, such as methotrexate, azathioprine, and cyclophosphamide. Since these drugs affect mitosis and cell division, they have severe toxic effects on normal cells with high turn-over rate such as bone marrow cells and the gastrointestinal tract lining. Accordingly, marrow depression and liver damage are common side effects.
Anti-inflammatory compounds used to induce immunosuppression include adrenocortical steroids such as dexamethasone and prednisolone. The common side effects observed with the use of these compounds are frequent infections, abnormal metabolism, hypertension, and diabetes.
Other immunosuppressive compounds currently used to inhibit lymphocyte activation and subsequent proliferation include cyclosporine, tacrolimus and rapamycin. Cyclosporine and its relatives are among the most commonly used immunosuppressant drugs. Cyclosporine is typically used for preventing or treating organ rejection in kidney, liver, heart, pancreas, bone marrow, and heart-lung transplants, as well as for the treatment of autoimmune and inflammatory diseases such as Crohn's disease, aplastic anemia, multiple-sclerosis, myasthenia gravis, uveitis, biliary cirrhosis, etc. However, cyclosporines suffer from a small therapeutic dose window and severe toxic effects including nephrotoxicity, hepatotoxicity, hypertension, hirsutism, cancer, and neurotoxicity.
Additionally, monoclonal antibodies with immunosuppressant properties, such as OKT3, have been used to prevent and/or treat graft rejection. Introduction of such monoclonal antibodies into a patient, as with many biological materials, induces several side-effects, such as dyspnea. Within the context of many life-threatening diseases, organ transplantation is considered a standard treatment and, in many cases, the only alternative to death. The immune response to foreign cell surface antigens on the graft, encoded by the major histo-compatibility complex (hereinafter referred as MHC) and present on all cells, generally precludes successful transplantation of tissues and organs unless the transplant tissues come from a compatible donor and the normal immune response is suppressed. Other than identical twins, the best compatibility and thus, long term rates of engraftment, are achieved using MHC identical sibling donors or MHC identical unrelated cadaver donors. However, such ideal matches are difficult to achieve. Further, with the increasing need of donor organs an increasing shortage of transplanted organs currently exists. Accordingly, xenotransplantation has emerged as an area of intensive study, but faces many hurdles with regard to rejection within the recipient organism.
The host response to an organ allograft involves a complex series of cellular interactions among T and B lymphocytes as well as macrophages or dendritic cells that recognize and are activated by foreign antigen. Co-stimulatory factors, primarily cytokines, and specific cell-cell interactions, provided by activated accessory cells such as macrophages or dendritic cells are essential for T-cell proliferation. These macrophages and dendritic cells either directly adhere to T-cells through specific adhesion proteins or secrete cytokines that stimulate T-cells, such as IL-12 and IL-15. Accessory cell-derived co-stimulatory signals stimulate activation of interleukin-2 (IL-2) gene transcription and expression of high affinity IL-2 receptors in T-cells. IL-2 is secreted by T lymphocytes upon antigen stimulation and is required for normal immune responsiveness. IL-2 stimulates lymphoid cells to proliferate and differentiate by binding to IL-2 specific cell surface receptors (IL-2R). IL-2 also initiates helper T-cell activation of cytotoxic T-cells and stimulates secretion of interferon-γ which in turn activates cytodestructive properties of macrophages. Furthermore, IFN-γ and IL-4 are also important activators of MHC class II expression in the transplanted organ, thereby further expanding the rejection cascade by enhancing the immunogenicity of the grafted organ The current model of a T-cell mediated response suggests that T-cells are primed in the T-cell zone of secondary lymphoid organs, primarily by dendritic cells. The initial interaction requires cell to cell contact between antigen-loaded MHC molecules on antigen-presenting cells (hereinafter referred as APC) and the T-cell receptor/CD3 complex on T-cells. Engagement of the TCR/CD3 complex induces CD154 expression predominantly on CD4 T-cells that in turn activate the APC through CD40 engagement, leading to improved antigen presentation. This is caused partly by upregulation of CD80 and CD86 expression on the APC, both of which are ligands for the important CD28 co-stimulatory molecule on T-cells. However, engagement of CD40 also leads to prolonged surface expression of MHC-antigen complexes, expression of ligands for 4-1BB and OX-40 (potent co-stimulatory molecules expressed on activated T-cells). Furthermore, CD40 engagement leads to secretion of various cytokines (e.g., IL-12, IL-15, TNF-α, IL-1, IL-6, and IL-8) and chemokines, all of which have important effects on both APC and T-cell activation and maturation. Similar mechanisms are involved in the development of auto-immune disease, such as type I diabetes. In humans and non-obese diabetic mice, insulin-dependent diabetes mellitus results from a spontaneous T-cell dependent auto-immune destruction of insulin-producing pancreatic .beta. cells that intensifies with age. The process is preceded by infiltration of the islets with mononuclear cells (insulitis), primarily composed of T lymphocytes. A delicate balance between auto-aggressive T-cells and suppressor-type immune phenomena determine whether expression of auto-immunity is limited to insulitis or not. Therapeutic strategies that target T-cells have been successful in preventing further progress of the auto-immune disease. These include neonatal thymectomy, administration of cyclosporine, and infusion of anti-pan T-cell, anti-CD4, or anti-CD25 (IL-2R) monoclonal antibodies. The aim of all rejection prevention and auto-immunity reversal strategies is to suppress the patient's immune reactivity to the antigenic tissue or agent, with a minimum of morbidity and mortality. Accordingly, a number of drugs are currently being used or investigated for their immunosuppressive properties. As discussed above, the most commonly used immunosuppressant is cyclosporine, which however has numerous side effects. Accordingly, in view of the relatively few choices for agents effective at immunosuppression with low toxicity profiles and manageable side effects, there exists a need in the art for identification of alternative immunosuppressive agents and for agents acting as complement to calcineurin inhibition.
There is also a need in the art to improve therapeutic efficiency by providing pharmaceutical compositions or combined preparations exhibiting a synergistic effect as a result of combining two or more immunosuppressant drugs, or antineoplastic drugs or anti-viral drugs.